Antibacterial and Antifungal Terpenes from the Medicinal Angiosperms of Asia and the Pacific: Haystacks and Gold Needles

This review identifies terpenes isolated from the medicinal Angiosperms of Asia and the Pacific with antibacterial and/or antifungal activities and analyses their distribution, molecular mass, solubility, and modes of action. All data in this review were compiled from Google Scholar, PubMed, Science Direct, Web of Science, ChemSpider, PubChem, and library searches from 1968 to 2022. About 300 antibacterial and/or antifungal terpenes were identified during this period. Terpenes with a MIC ≤ 2 µg/mL are mostly amphiphilic and active against Gram-positive bacteria, with a molecular mass ranging from about 150 to 550 g/mol, and a polar surface area around 20 Å². Carvacrol, celastrol, cuminol, dysoxyhainic acid I, ent-1β,14β-diacetoxy-7α-hydroxykaur-16-en-15-one, ergosterol-5,8-endoperoxide, geranylgeraniol, gossypol, 16α-hydroxy-cleroda-3,13 (14)Z-diene-15,16-olide, 7-hydroxycadalene, 17-hydroxyjolkinolide B, (20R)-3β-hydroxy-24,25,26,27-tetranor-5α cycloartan-23,21-olide, mansonone F, (+)-6,6′-methoxygossypol, polygodial, pristimerin, terpinen-4-ol, and α-terpineol are chemical frameworks that could be candidates for the further development of lead antibacterial or antifungal drugs.

The outer structure of bacteria and fungi provides resistance to terpenes and other xenobiotics. Gram-negative bacteria, compared to Gram-positive bacteria, are more resistant to plant natural products and antibiotics because they are packed in a hydrophilic and

Sesquiterpenes
MIC are listed in Table S1.

Diterpenes
MIC are listed in Table S1.

Diterpenes
MIC are listed in Table S1.

Cyclic Diterpenes
The cyclization of geranylgeranyl diphosphate accounts for the formation of all antibacterial and antifungal cyclic diterpenes ( Figure 6).

Triterpenes
MIC are listed in Table S1. The condensation of a pair of farnesyl cations forms 2.3-oxidosqualene, from which all antibacterial and antifungal triterpenes are derived by cyclisation (Figure 7).

The Distribution of Antibacterial and Antifungal Terpenes
Regarding the distribution of antibacterial and antifungal terpenes among Asian medicinal Angiosperms, it can be seen in Table 1 that all clades, except the Rosids, yield antibacterial and/or antifungal terpenes. Clades in the Core Eudicots tend to synthesise specific classes of antibacterial and/or antifungal terpenes such as dihydroagarofurans, jatrophanes, cassanes, and cucurbitanes (Fabids), or santalanes, quassinoids, and limonoids (Malvids). The Malvids are home to the broadest array of antibacterial and antifungal sesquiterpenes and triterpenes. In the Upper Angiosperms, Lamiids bring to being the broadest array of antibacterial and antifungal diterpenes. Antibacterial and antifungal terpenes with MIC ≤2 µg/mL are produced by plants in all three groups of Angiosperms.
For terpenes liquid at room temperature, we suggest very strong activity for a value below or equal to 2 µL/mL. According to Tampieri et al. (2005), strong activity is defined for natural products with MIC values ≤ 50 ppm [16]. Here, a terpene is defined as having moderate activity for MIC > 50 and ≤ 100 ppm; weak activity for MIC > 100 and ≤1500 ppm and inactivity for MIC > 1500 ppm.
Accordingly, out of about 300 antibacterial and/or antifungal terpenes identified between 1968 and 2022, 18 (four monoterpenes, five sesquiterpenes, four diterpenes, and five triterpenes) exhibited very strong activities ( Table 2). Most of these were active against Gram-positive bacteria, followed by Gram-negative bacteria, mycobacteria, filamentous fungi, and yeasts.

Influence of Molecular Mass
The molecular mass of natural products influences their ability to fit in the catalytic pockets of enzymes, cytoplasmic membrane, and to cross the outer membrane via porins. Here, a low molecular mass was defined as below 200 g/mol, medium molecular mass from 200 to 400 g/mol, and high molecular mass above 400 g/mol. Following this classification, terpenes with MIC ≤ 2 µg/mL have a molecular mass ranging mainly from about 150 to 550 g/mol ( Table 2).
Terpenes with low molecular mass are active against both Gram-positive and Gramnegative bacteria. It can be argued that being volatile, monoterpenes evaporate from paper discs or agar wells or even liquid broths explaining low activities recorded by most authors, except for Orhan et al., using emulsions [15,186]. Furthermore, the determination of MIC in the liquid broth of non-polar terpenes is almost impossible because they do not dissolve in an aqueous broth and we suggest using paper discs or dissolving the terpenes in melted solid agar for test in Petri dishes for this purpose as well as for measuring synergistic activities. Dimethyl sulfoxide has been recommended to facilitate the dissolution of nonpolar natural products in liquid broth, but it has antibacterial and cytotoxic effects and does not dissolve most non-polar extracts and terpenes (personal communication).
Medium molecular mass is beneficial for activity against yeasts whereas filamentous fungi are sensitive to terpenes with low, medium, and high molecular masses.
Six out of the 18 terpenes with MIC ≤ 2 µg/mL had a high molecular mass and terpenes with a high molecular mass were only active against Gram-positive bacteria, probably because of their inability to cross porin channels. It can be observed that terpenes with strong activity against mycobacteria have medium to high molecular masses.

Influence of Solubility and Polar Surface Area
Water-soluble and amphiphilic terpenes cross porin channels [4]. Here, we define (at pH 7.4) terpenes with LogD below 1 as hydrophilic, LogD between 1 and 5 amphiphilic, andLogD above 5 as liposoluble.
Accordingly, it can be observed in Table 2 that there are no hydrophilic terpenes with MIC < 2 µg/mL. Amphiphilic terpenes are active against both Gram-positive and Gramnegative bacteria. Lipophilic terpenes are active against Gram-positive bacteria, specifically against mycobacteria, as they might dissolve into mycolic acid. The solubility of terpenes does not influence the activity against filamentous fungi whereas yeasts are specifically sensitive to mid-polar terpenes. The polar surface area of terpene with very strong activity is around 20 Å 2 .

Structure Activity and Mechanism of Action
Regarding the structure-activity relationship and mode of action of terpenes, a general observation is that aromaticity, planarity, and substitutions with hydroxyl, ketone, aldehyde, or carboxylic acid groups increase the antibacterial and antifungal activities of terpenes. The presence of peroxide and/or epoxide groups is beneficial for antibacterial and antifungal properties as seen is amblyone (89), 1,8-cineole (27), artemisinin (60), 17hydroxyjolkinolide B (68), or ergosterol-5,8-endoperoxide (107). Lipophilic terpenes are often antimycobacterial [96].
Linear monoterpenes inhibit the growth of both Gram-positive and Gram-negative bacteria, suggesting the targeting of the cytoplasmic and/or outer membrane. They are ineffective against Mycobacteria. Cyclic monoterpenes are broad-spectrum antibacterial and antifungal, but not antimycobacterial. The antibacterial mode of action of monoterpenes invokes the destabilisation of the cytoplasmic membrane such as in α-terpineol (24), terpinen-4-ol (25), δ-terpineol (26), and 1, 8-cineole (27). Specific mechanisms arise with aromaticity, as seen with thymoquinone (36), which inhibits E. coli ATP synthase and in C. albicans induces the generation of reactive oxygen species [32,33,37,43,[215][216][217]. The antifungal mode of action of monoterpenes includes cytoplasm coagulation, hyphal lysis, cell membrane insults, and the leakage of cellular cytoplasmic components [34]. The fact that the reduction and oxidation or isomerization of monoterpene does not much influence their strength against a broad-spectrum of bacteria and fungi spectrum points to mainly non-specific mechanisms, and most probably, accumulation in and the destabilisation of cytoplasmic membranes.
Sesquiterpenes are mainly broad-spectrum antibacterial, antimycobacterial, and antifungal via a mix of non-specific and specific mechanisms. Non-substituted sesquiterpenes like α-humulene (61) non-specifically target the membrane of Gram-positive bacteria and increase the permeability and intracellular content leakage [217]. The cytoplasmic membrane is also one of the non-specific fungal targets of amphiphilic sesquiterpenes, as seen with polygodial (64) with S. cerevisiae [91] and cadinanes [73]. For linear sesquiterpenes, the oxidation of hydroxyl groups into aldehyde is detrimental for activity against filamentous fungi. The introduction of a lactone moiety in sesquiterpenes boosts their activity against filamentous fungi, as seen with costunolide (45), cynaropicrin (46), deacetylxanthumine (47), and isoalantolactone (48) [47]. α-Methylene lactone moieties open to form Michaeltype amine adducts with bacterial and fungal amino acids and ribonucleic acids. Furanone moieties in the presence of metal ions generate reactive oxygen species, forming strand breaks and the formation of 8-hydroxy-2 -deoxyguanosine in microbial DNA. Planarity and aromaticity translate into strong antibacterial (Gram-positive) properties, as seen with mansonone F (56) [71] and gossypol (52), the latter targeting DNA polymerase [218][219][220][221]. Epoxide groups are favourable for activity against Gram-negative bacteria, as seen with artemisinin (60), via copper-mediated DNA damage [79].
Triterpenes are active against Gram-positive and Gram-negative bacteria, mycobacteria, yeasts, and filamentous fungi. The mechanism of action of triterpenes involves both non-specific and specific mechanisms. Lipophilic or amphiphilic triterpenes tend to damage the membrane with subsequent leakage of intracellular K + , as seen with geranylgeraniol (65) and (E)-phytol (66) [95,222]. Triterpenes with benzoquinone moieties, the ketone moiety in ring A conjugated with double bonds and substitution with carboxylic acid groups are strongly active [145] and tend to target bacterial and fungal DNA and/or topoisomerases, as seen with celastrol (98) and zeylasterone (99) [175,176]. Zeylasterone (99) induces cell membrane alterations in B. subtilis [176]. Limonoids inhibit DNA polymerase [223]. An increase in the lipophilicity and presence of endoperoxide or epoxide groups are beneficial for antimycobacterial and anti-Gram-negative activities, as seen with epoxy dammaranes [141]. The catabolism of cholesterol in M. tuberculosis requires enzymes [197] targeted by triterpenes and steroids. Triterpene saponins tend to target Gram-positive bacterial surface sortases [198], and like dioscin (109), lethal for C albicans via the formation of complexes with ergosterol in the cell membrane of fungi leading to the formation of pores, the loss of membrane integrity, and the leakage of cytoplasmic content [198,199,205,206,224,225].
Specific mechanisms: Antibacterial potentiators such as clerodanes [229], carnosic acid (70), and oleananes [230] inhibit bacterial and fungal efflux pumps. Tiglianes inhibit Pglycoprotein in HepG2/ADR cells, and as such, might be able to inhibit bacterial and/or fungal efflux pumps [231]. Clerodanes inhibit NorA efflux pumps in S. aureus [232]. Neuroactive terpenes tend to inhibit bacterial efflux pumps. An example of neuroactive natural products inhibiting bacterial NorA is the monoterpene indole alkaloid reserpine from Rauvolfia serpentina (L.) Benth. ex Kurz (Apocynaceae; Lamiids). Additionally, reserpine is a calcium channel antagonist as is the synthetic calcium channel antagonist verapamil [233][234][235][236]. The reason why the calcium channel antagonists inhibit the bacterial efflux pump is, at least in part, because of the correlations between the bacterial efflux pumps and bacterial calcium transport [237]. Specific potentiators interfere with the cytoplasmic membrane polarisation of bacteria or fungi, resulting in efflux pump inhibition, as seen with cardenolides [238] and sesquiterpene lactones [239]. Another interesting feature of terpenes, and especially diterpenes, is their ability to remove genes of resistance from the plasmids of Gram-negative bacteria [122].

The Safety Issues of Terpenes with Respect on Human Health
Terpenes are phytoalexins/phytoanticipins produced by plants to poison/repel microbes, other plants, and animals [240]. For instance, mansonone E is antifeedant and phytotoxic [68]. In humans, terpenes can induce allergies, irritations as well as renal, pulmonary, hepatic, neurological, or cardiovascular damage [241][242][243]. Cardenolides are cardiotoxic, and euphorbiaceous phorbol esters are tumorigenic. At the cellular level, toxic terpenes disrupt cytoplasmic membranes, generate reactive oxygen species, and impair mitochondrial function [244]. Planar terpenes targeting bacterial DNA are often cytotoxic [66] as well as jatrophanes, daphnanes [130], gypsogenin [155], quassinoids [191], and steroidal saponins [207]. Therefore, selectivity indices using mammalian cells in vitro or lethal doses 50% (LD 50 ) in studies using rodent are advised. The use of brine shrimps (Artemia salina) to determine the toxicity of antimicrobial terpenes is very simple and inexpensive [143]. Weinstein and Albersheim (1983) argue that antibacterial natural products from plants act via non-specific mechanisms preventing the development of resistance [245]. The medicinal Angiosperms of Asia and the Pacific generate an enormous diversity of antibacterial and antifungal terpenes acting via specific and/or non-specific mechanisms representing a vast source of potential antimicrobial leads. However, terpenes are often difficult to isolate and identify, tend not to have good oral bioavailability, and are often toxic. For these reasons, identifying antibacterial or antifungal terpenes of clinical systemic usefulness is like trying to find a few needles in a large haystack, but the search is worthwhile.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/molecules28093873/s1, Table S1: Antibacterial and antifungal terpenes from the medicinal plants of Asia and the Pacific.